The importance of alternative RNA splicing in the generation of genetic diversity is now widely recognized as one of the most important ways (along with the use of alternative promoters and alternative polyadenylation) for a single gene to encode more than one mRNA transcript. The pre-mRNA or mRNA isoforms that result from alternative splicing may differ in stability, translatability, or protein sequence encoded, each of which may alter the function of the encoded protein.
This can be best exemplified in the field of apoptosis (Jiang and Wu., Proc. Soc. Exp. Biol. Med. 220: 64-72 (1999)). Alterations in alternative splicing, in particular mutations of the canonical sequences at the intron/exon border, may cause abnormal splicing patterns that affect gene expression and cause disease. Cooper et al. (1998) recently showed that at least 10% of human inherited diseases involve mutations that create an RNA splicing defect; see e.g., Cooper et al., Nucleic Acids Res. 26: 285-287 (1998).
Sporadic mutations in the consensus splicing signals are observed in a wide-range of pathologies such as cancers, neurodegenerative disorders, inflammation/asthma and other metabolic diseases. The RNA splicing defects can include exon skipping, intron retention and new splicing events due to the use of cryptic splicing sites or the creation of new splicing consensus sequences (Lopez, Annu. Rev. Genet. 32: 279-305 (1998)). Alterations in activity, levels, or amino acid sequence of cellular splicing factors may affect the efficiency of splicing or the regulation of alternative splicing. For example, the presence of a single nucleotide in the nucleotide sequence of the Survival Motor Neuron gene regulates the splicing of this gene, and is responsible for spinal muscular atrophy (Lorson et al. Proc. Natl. Acad. Sci. USA, 96: 6307-6311 (1999)). In human brains taken from patients with sporadic Alzheimer's disease, splicing events including a) alterations in the amino acid sequence of the protein presinilin-1 (PS1), caused by a deletion of exon 9 of the ps1 gene, b) deletion of exon 5 of the gene encoding presenilin 2 (the ps2 gene), and c) (in cases of sporadic frontotemporal dementia) aberrant splicing of exon 5 of the ps2 gene have been be implicated in the neuropathology (Isoe-Wada. et al. Eur J Neurol, 6, 163-167 (1999); Sato et al. J. Biol. Chem. 276: 2108-2114 (1991)).
Alzheimer's disease (AD) is a devastative degenerative disorder of the brain with important formation of amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss (Hardy et al. Nat. Neurosci 1:355-358 (1998); Selkoe, D. J. In: Alzheimer disease, Ed 2 (Terry, R. D., Katzman, R., Bick, K. L., Sisodia, S. S., eds), pp 293-310. Philadelphia: Lippincott Williams and Wilkins. (1999)). The most affected regions are cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. Patients suffering from AD have increased problems with memory loss, intellectual functions and skills, personality changes and schizophrenia. AD is the leading cause of dementia in elderly persons and there is no effective palliative or preventive treatment for the neurodegeneration.
Several genetic and epigenetic factors have been suggested as mechanisms contributing to AD; these include genetic predisposition, infectious agents, toxins, metals, head trauma and vascular dementia. Globally, it is the dysregulation of intracellular pathways responsible for amyloid precursor protein (APP) proteolytic processing that results in enhanced formation of a peptide termed A-Beta (A-β) 1-42—a form of the A-β peptide which is particularly amyloidogenic, which now appears to be central to the pathophysiology of AD (Selkoe, Neuron 32: 177-180 (2001).
The A-β peptide is also the primary protein constituent in cerebrovascular amyloid deposits. Amyloid is a filamentous material that is arranged in beta-pleated sheets. The A-β peptide is a hydrophobic peptide comprising up to 43 amino acids. A-β peptide has been shown to be toxic to neurons in a number of ways, including by the induction of reactive oxygen species (ROS), induction of altered gene transcription, causing increased susceptibility to excitotoxicity, and other processes commonly associated with neurodegenerative conditions ((Ramsden et al., J. Neurochem. 79: 699-712 (2001); Shukla et al., J. Cell. Path. 5: 241-249 (2002); Green and Peers, Neurochem. 77: 953-956 (2001); Kowall et al., Neurobiol. Aging 13: 537-542 (1992); MacManus et al., J. Biol. Chem. 275: 4713-4718 (2000)). Mutations in APP, Presenilin 1 and 2 (PS1 and PS2, respectively) greatly alter APP processing, resulting in enhanced A-β 1-42 formation. Amyloid plaques are also detected in aged patients with Down's Syndrome who survive up to the age of 30. The observed up-regulation of APP expression in Down's Syndrome is probably a cause of the development of AD in Down's patients (Rumble et al., N. Engl. J. Med. 320:1446-52 (1989); Mann, Neurobiol. Aging 10: 397-399 (1989)). Amyloid plaques are also present in the normal aging brain, although at a lower number (Vickers et al., Exp. Neurol. 141:1-11 (1996)).
The different forms of human APP presently known range in size from 695-770 amino acids, localize to the cell surface, and have a single C-terminal transmembrane domain. A number of APP cDNA's have been identified, including the three most abundant forms, APP695 described by Kang et al. (1987) Nature 325: 733-736 which is designated as the “normal” APP; the 751 amino acid polypeptide (APP751) described by Tanzi et al. (1988) Nature 331: 528-530; and the 770 amino acid polypeptide (APP770) described by Kitaguchi et. al. (1988) Nature 331: 530-532. These forms arise from a single precursor RNA by alternative splicing. The A-β peptide, which is common to each of the three splice variants of APP, is derived from a region of APP adjacent to and containing a portion of the transmembrane domain.
Three different proteases process APP in vivo (Vassar and Citron, Neuron 27: 419-422 (2000)). Alpha-secretase cleaves APP 12 amino acid residues from the lumenal surface of the plasma membrane; it is not involved in A-β production. The first step of Aβ generation is performed by cleavage of APP by β-secretase (BACE), a type I membrane-bound aspartyl protease. BACE cleavage generates a 100-kDa soluble form (sAPP) of the ectodomain—the portion of APP that projects from the cell surface—and a 12-kDa membrane-associated intermediate peptide of 99 amino acids (termed C99) containing the N-terminus of the AB peptide (Vassar et al., Science 286(5440):735-41 (1999). The C99 peptide is then processed by the protease gamma-secretase to yield various Aβ peptides differing in size or terminal modification (40-42 and 43 amino acid residues being the most frequent peptides found in vivo) (for review, see Selkoe et al., Nature 399(6738 Suppl):A23-31 (1999); Tekirian, J. Alzheimers. Dis. 3(2):241-248. (2001)). The APP sequence near the β-secretase cleavage site is:                EVKM*DAE. (SEQ ID NO: 34)        
These residues are labeled P4-P3-P2-P1*P1′-P2′-P3′ in standard protease nomenclature with the cleavage site between P1 and P1′ marked by *. Mutations in this region, such as the KM to NL mutation (the so-called Swedish mutation), can transform APP into a more preferred substrate for BACE. Hence, amino acid sequence changes in APP that result in increased APP cleavage by BACE increase the likelihood of the development of Alzheimer's (Citron et al., Nature 360(6405): 672-674 (1992)). Experimental evidence suggests that APP processing is sequential and that cleavage of APP by beta-secretase is a prerequisite for gamma-secretase-mediated APP processing. Cleavage within the transmembrane region of APP by gamma-secretase results in the 40/42-residue Aβ peptide, whose elevated production and accumulation in the brain are the central events in the pathogenesis of Alzheimer's disease (Selkoe,. Nature 399:23-31 (1999)). In addition, it is now clear that BACE can again cut Aβ peptide 40-42 after gamma-secretase to generate a neurotoxic Aβ34 peptide, at the expense of Abeta40-42 (Fluhrer et al., J. Biol. Chem. 278(8): 5531-5538 (2003)).
Many of the existing therapeutic strategies for AD have focused on gamma-secretase inhibition. However, it now appears that such strategies may not be sufficient, or even sound, to treat or prevent AD. For example, it is now clear that the C99 peptide itself, which requires BACE and not gamma-secretase cleavage for generation, includes the entire Aβ peptide, and is neurotoxic when evaluated in cultured cells, also accumulates in the AD brain (Tekirian, J. Alzheimers. Dis. 3(2):241-248. (2001)). Furthermore, gamma-secretase inhibitors have been shown to seriously affect the immune system and result in the accumulation of C terminal APP fragments, which are themselves toxic. In addition, gamma-secretase inhibition may alter the processing of various vital proteins (Doerfler, et al., Proc. Natl. Acad. Sci. USA 98 :9312-9317 (2001), Ni et al., Science 294: 2179-2181 (2001), Marambaud, et al., EMBO J. 21: 1948-1956 (2002), Kim, et al., J. Biol. Chem. 277: 499976-499981 (2002)). While a lack of BACE activity is not inevitably fatal in utero, a double-genetic knock-out of the presenilin 1 and 2 genes did prove to be so (Herreman, et al. Proc. Natl. Acad. Sci. USA 96: 11872-11877 (1999)). This toxicity is primarily a result of inhibition of Notch signaling pathway, which is involved in cell-to-cell signaling. Indeed, the Notch receptor and its cognate ligands Jagged and Delta are known substrates of presenilins (De Strooper, et al. Nature 398: 518-522 (1999)) and because the cleavage of Notch and its ligands leads to the release of proteolytic fragments active in cell signaling (LaVoie and Selkoe, J. Biol. Chem. 278(36): 34427-34437 (2003)).
In contrast, the in-vivo processing of the β-secretase site of the APP peptide is thought to be the rate-limiting step in Aβ peptide production (Sinha, S. & Lieberburg, Proc Natl Acad Sci USA. 96(20):11049-53 (1999); Vassar, Adv. Drug Deliv. Rev. 54(12):1589-1602 (2002)). BACE does not appear to participate in the generation of other physiologically important proteins (Cai et al., Nat. Neurosci 4: 233-234 (2001); Luo et al., Nat. Neurosci 4: 231-232 (2001)). Therefore, BACE appears as the strongest therapeutic target for decreasing or inhibiting Aβ generation.
BACE is synthesized as an inactive pro-enzyme. During maturation in the secretory pathway, BACE undergoes glycosylation at 3 of 4 N-linked sites and is separated from its propeptide domain by a member of the proprotein convertase family of proteases. In addition, BACE also undergoes palmitoylation at cysteine residues within its cytoplasmic domain and is phosphorylated at its C terminus. After core glycosylation in the endoplasmic reticulum (ER), BACE is rapidly and efficiently transported to the Golgi apparatus before targeting to the endosomal system (Fluhrer, R. et al.; J Biol. Chem. 278(8):5531-5538 (2003)).
To date, three isoforms of BACE have been isolated: BACE476, BACE457 and BACE432. All of these isoforms are in-frame deletions generated by the alternative usage of the exon3 region of the gene, and all of them exhibit much less APP processing activity, if any at all, than BACE501 (Bodendorf, et al., J. Biol. Chem. 276(15):12019-12023 (2001), Zohar et al., Brain Res. Mol. Brain. Res. 115(1):63-68 (2003), Tanahashi and Tabira, Neurosci. Lett. 307(1):9-12 (2001)).